Project Summary gd T cells remain an enigma of the immune system. Much of this results from an ignorance of the ligands for gd T cells. gd T cells often localize at epithelial barriers, sites of inflammation with infection, autoimmunity, or tumors, and appear to promote tissue homeostasis at these sites. Consistect with this, a recent seminal study reported that intratumor gd T cells are the most favorable prognostic immune population across 39 cancer types in humans. However, compared to ab T cells, almost nothing is known regarding the specificity of gd T cells or the structure of the TCR-gd complexed with its ligands. From our studies in human Lyme and rheumatoid arthritis we have developed a panel of synovial Vd1 gd T cell clones. From three of these we cloned the TCR-gd and produced a soluble TCR-gd (sTCR-gd) that was tetramerized for flow cytometry. This reagent revealed a broad spectrum of staining of a panel of 15 tumor cell lines, as well as activated monocytes and T lymphocytes. The staining patterns were combined with RNAseq data on the same tumor lines and bioinformatics performed to narrow the list of candidate ligands. We also determined that the ligand(s) is a protein that contains needed glycosaminoglycans (GAGs). This was confirmed by both enzymatic digestion of GAGs and CRISPR/Cas9 deletion of a sulfate transporter the promotes GAG formation. We have also produced initial crystals of the sTCR-gd for eventual structure analysis, and lentivirally transfected the TCR-gd into TCR-negative Jurkat T cells. The Specific Aims are thus: Specific Aim 1. Identify synovial TCR-gd candidate ligands using RNAseq and bioinformatics. We will expand the number of tumor lines examined to the full panel of 24 cell lines for which RNAseq data is available. Similar analyses will be performed on activated monocytes and T cells. This will increase the power of the bioinformatics analysis. Specific Aim 2. Confirm TCR-gd candidate ligands and pathways of synthesis using CRISPR/Cas9. We will perform CRISPR/Cas9 of targeted candidate ligands from Aim 1. In parallel, we will perform an independent genome-wide CRISPR/Cas9 screen to also identify molecules and pathways involved with ligand synthesis This project combines the expertise of three co-PIs: Dr. Ralph Budd in human synovial gd T cell biology, Dr. Simon Davis in novel bioinformatics approaches to identifying cell surface ligands for orphan receptors, and Dr. Gavin Wright in genome-wide CRISPR/Cas9 analyses. It also uses novel reagents, being the only laboratory that has human syonovial gd T cell clones, and unique soluble TCR-gd from three clones. Furthermore, all the required technqiues are in place, as are the collaborators. The findings would provide an enormous advance for human gd T cell biology, and enable future studies on the regulation of TCR-gd ligands as well as the structure of a synovial TCR-gd in complex with its ligand.